Chromatin Immunoprecipitation for Transcription Factors

Chromatin was prepared from the indicated T helper cell population as previously described^{17 (link)}. The resulting chromatin was incubated with either anti-STAT4 (Santa Cruz), anti-STAT3 (Santa Cruz), or control antibody (Abcam) and immunoprecipitated using Protein G Dynabeads (Life Technologies). Precipitated DNA was analyzed via quantitative PCR using SYBR Select Mastermix (Life Technologies) and gene-specific primers: (Bcl6 promoter forward: 5′-GCGGAGCAATGGTAA AGCCC-3′, and reverse: 5′-CTGGTGTCCGGCCTTTCCTAG-3′; Bcl6 control forward: 5′-GTACTCCAACAACAGCACAGC-3′, and reverse: 5′-GTGGCTCGTTAAATCACAGAGG-3′; Il21 promoter forward: 5′-CAC ACACCTTGGTGAATGCTG-3′, and reverse: 5′-CCATTGGCTAGGTGTACGTGTG-3′). Samples were normalized to total input followed by the subtraction of the isotype control to account for unspecific binding.